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. 1996 Nov;70(11):8138–8141. doi: 10.1128/jvi.70.11.8138-8141.1996

Novel retroviral vector transferring a suicide gene and a selectable marker gene with enhanced gene expression by using a tetracycline-responsive expression system.

J J Hwang 1, Z Scuric 1, W F Anderson 1
PMCID: PMC190890  PMID: 8892941

Abstract

A retroviral vector for the enhanced expression of the herpes simplex virus thymidine kinase (HSV tk) gene was developed by using a tetracycline-responsive expression system (TRES). The two components of the TRES, the chimeric transactivator (tTA) and the corresponding tTA-binding cis element (tetO), were both incorporated into a retroviral vector and resulted in high levels of tk gene expression from tetO in target cells. Amphotropic virus supernatants from stable producer cells, generated by the retroviral vector containing the TRES, gave titers of 10(4) to 10(5) G418-resistant CFU/ml on murine NIH 3T3 cells. The retroviral vector (G1tTA-[tetOTkINa]R), in which tetO was used in the opposite orientation relative to viral transcription, was capable of transducing tk and neo genes into murine NIH 3T3 cells to yield a high level of tk gene expression. TK enzyme activity in NIH 3T3 cells transduced by this vector was 417-fold higher than in control cells. This increased TK activity was returned to basal levels in the presence of tetracycline. The level of tk gene expression driven by tetO from G1tTA-[tetOTkINa]R vector in NIH 3T3 cells was fourfold higher at both the mRNA level and the TK enzyme level than that produced by the long terminal repeat of G1Tk1SvNa, the vector being used in the ongoing brain tumor gene therapy trial. Retroviral vectors containing the TRES may be useful therefore in achieving higher levels of tk gene expression, which should facilitate gene therapy approaches in the treatment of cancer.

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Selected References

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  1. Anderson W. F. Human gene therapy. Science. 1992 May 8;256(5058):808–813. doi: 10.1126/science.1589762. [DOI] [PubMed] [Google Scholar]
  2. Chen C. Y., Chang Y. N., Ryan P., Linscott M., McGarrity G. J., Chiang Y. L. Effect of herpes simplex virus thymidine kinase expression levels on ganciclovir-mediated cytotoxicity and the "bystander effect". Hum Gene Ther. 1995 Nov;6(11):1467–1476. doi: 10.1089/hum.1995.6.11-1467. [DOI] [PubMed] [Google Scholar]
  3. Dhawan J., Rando T. A., Elson S. L., Bujard H., Blau H. M. Tetracycline-regulated gene expression following direct gene transfer into mouse skeletal muscle. Somat Cell Mol Genet. 1995 Jul;21(4):233–240. doi: 10.1007/BF02255778. [DOI] [PubMed] [Google Scholar]
  4. Duch M., Paludan K., Jørgensen P., Pedersen F. S. Lack of correlation between basal expression levels and susceptibility to transcriptional shutdown among single-gene murine leukemia virus vector proviruses. J Virol. 1994 Sep;68(9):5596–5601. doi: 10.1128/jvi.68.9.5596-5601.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Eglitis M. A., Anderson W. F. Retroviral vectors for introduction of genes into mammalian cells. Biotechniques. 1988 Jul-Aug;6(7):608–614. [PubMed] [Google Scholar]
  6. Emerman M., Temin H. M. Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell. 1984 Dec;39(3 Pt 2):449–467. [PubMed] [Google Scholar]
  7. Emerman M., Temin H. M. Quantitative analysis of gene suppression in integrated retrovirus vectors. Mol Cell Biol. 1986 Mar;6(3):792–800. doi: 10.1128/mcb.6.3.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Flowers M. E., Stockschlaeder M. A., Schuening F. G., Niederwieser D., Hackman R., Miller A. D., Storb R. Long-term transplantation of canine keratinocytes made resistant to G418 through retrovirus-mediated gene transfer. Proc Natl Acad Sci U S A. 1990 Mar;87(6):2349–2353. doi: 10.1073/pnas.87.6.2349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Furth P. A., St Onge L., Böger H., Gruss P., Gossen M., Kistner A., Bujard H., Hennighausen L. Temporal control of gene expression in transgenic mice by a tetracycline-responsive promoter. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9302–9306. doi: 10.1073/pnas.91.20.9302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ghattas I. R., Sanes J. R., Majors J. E. The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos. Mol Cell Biol. 1991 Dec;11(12):5848–5859. doi: 10.1128/mcb.11.12.5848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gossen M., Bujard H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci U S A. 1992 Jun 15;89(12):5547–5551. doi: 10.1073/pnas.89.12.5547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hoshimaru M., Ray J., Sah D. W., Gage F. H. Differentiation of the immortalized adult neuronal progenitor cell line HC2S2 into neurons by regulatable suppression of the v-myc oncogene. Proc Natl Acad Sci U S A. 1996 Feb 20;93(4):1518–1523. doi: 10.1073/pnas.93.4.1518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Karlsson S., Papayannopoulou T., Schweiger S. G., Stamatoyannopoulos G., Nienhuis A. W. Retroviral-mediated transfer of genomic globin genes leads to regulated production of RNA and protein. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2411–2415. doi: 10.1073/pnas.84.8.2411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kay M. A., Baley P., Rothenberg S., Leland F., Fleming L., Ponder K. P., Liu T., Finegold M., Darlington G., Pokorny W. Expression of human alpha 1-antitrypsin in dogs after autologous transplantation of retroviral transduced hepatocytes. Proc Natl Acad Sci U S A. 1992 Jan 1;89(1):89–93. doi: 10.1073/pnas.89.1.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Levine F., Yee J. K., Friedmann T. Efficient gene expression in mammalian cells from a dicistronic transcriptional unit in an improved retroviral vector. Gene. 1991 Dec 15;108(2):167–174. doi: 10.1016/0378-1119(91)90431-a. [DOI] [PubMed] [Google Scholar]
  16. Lyons R. M., Forry-Schaudies S., Otto E., Wey C., Patil-Koota V., Kaloss M., McGarrity G. J., Chiang Y. L. An improved retroviral vector encoding the herpes simplex virus thymidine kinase gene increases antitumor efficacy in vivo. Cancer Gene Ther. 1995 Dec;2(4):273–280. [PubMed] [Google Scholar]
  17. McLachlin J. R., Mittereder N., Daucher M. B., Kadan M., Eglitis M. A. Factors affecting retroviral vector function and structural integrity. Virology. 1993 Jul;195(1):1–5. doi: 10.1006/viro.1993.1340. [DOI] [PubMed] [Google Scholar]
  18. Miller A. D., Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol. 1986 Aug;6(8):2895–2902. doi: 10.1128/mcb.6.8.2895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miller A. D. Human gene therapy comes of age. Nature. 1992 Jun 11;357(6378):455–460. doi: 10.1038/357455a0. [DOI] [PubMed] [Google Scholar]
  20. Miller A. D. Retrovirus packaging cells. Hum Gene Ther. 1990 Spring;1(1):5–14. doi: 10.1089/hum.1990.1.1-5. [DOI] [PubMed] [Google Scholar]
  21. Morgan R. A., Couture L., Elroy-Stein O., Ragheb J., Moss B., Anderson W. F. Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy. Nucleic Acids Res. 1992 Mar 25;20(6):1293–1299. doi: 10.1093/nar/20.6.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Osborne W. R. Retrovirus-mediated gene expression in mammalian cells. Curr Opin Biotechnol. 1991 Oct;2(5):708–712. doi: 10.1016/0958-1669(91)90039-8. [DOI] [PubMed] [Google Scholar]
  23. Palmer T. D., Rosman G. J., Osborne W. R., Miller A. D. Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1330–1334. doi: 10.1073/pnas.88.4.1330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Paulus W., Baur I., Boyce F. M., Breakefield X. O., Reeves S. A. Self-contained, tetracycline-regulated retroviral vector system for gene delivery to mammalian cells. J Virol. 1996 Jan;70(1):62–67. doi: 10.1128/jvi.70.1.62-67.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Salmons B., Günzburg W. H. Targeting of retroviral vectors for gene therapy. Hum Gene Ther. 1993 Apr;4(2):129–141. doi: 10.1089/hum.1993.4.2-129. [DOI] [PubMed] [Google Scholar]
  26. Shockett P., Difilippantonio M., Hellman N., Schatz D. G. A modified tetracycline-regulated system provides autoregulatory, inducible gene expression in cultured cells and transgenic mice. Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6522–6526. doi: 10.1073/pnas.92.14.6522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Williams D. A. Expression of introduced genetic sequences in hematopoietic cells following retroviral-mediated gene transfer. Hum Gene Ther. 1990 Fall;1(3):229–239. doi: 10.1089/hum.1990.1.3-229. [DOI] [PubMed] [Google Scholar]
  28. Wilson J. M., Chowdhury N. R., Grossman M., Wajsman R., Epstein A., Mulligan R. C., Chowdhury J. R. Temporary amelioration of hyperlipidemia in low density lipoprotein receptor-deficient rabbits transplanted with genetically modified hepatocytes. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8437–8441. doi: 10.1073/pnas.87.21.8437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yarranton G. T. Inducible vectors for expression in mammalian cells. Curr Opin Biotechnol. 1992 Oct;3(5):506–511. doi: 10.1016/0958-1669(92)90078-w. [DOI] [PubMed] [Google Scholar]

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